Malate Dehydrogenase Isoforms Research Articles

Malate dehydrogenase as a multi-purpose target for drug discovery.

Malate dehydrogenase (MDH) enzymes play critical roles in cellular metabolism, facilitating the reversible conversion of malate to oxaloacetate using NAD+/NADH as a cofactor. The two human isoforms of MDH have roles in the citric acid cycle and the malate-aspartate shuttle, and thus both are key enzymes in aerobic respiration as well as regenerating the pool of NAD+ used in glycolysis. This review highlights the potential of MDH as a therapeutic drug target in various diseases, including metabolic and neurological disorders, cancer, and infectious diseases. The most promising molecules for targeting MDH have been examined in the context of human malignancies, where MDH is frequently overexpressed. Recent studies have led to the identification of several antagonists, some of which are broad MDH inhibitors while others have selectivity for either of the two human MDH isoforms. Other promising compounds have been studied in the context of parasitic MDH, as inhibiting the function of the enzyme could selectively kill the parasite. Research is ongoing with these chemical scaffolds to develop more effective small-molecule drug leads that would have great potential for clinical applications.

PECULIARITIES OF MALATE DEHYDROGENASE ISOENZYME COMPOSITION AND ENZYMATIC ACTIVITY IN AMARANTH VARIETIES AND VARIETAL POPULATIONS OF DIFFERENT RIPENESS GROUPS

The article presents the results of studying the enzymatic activity of malate dehydrogenase in amaranth varieties and breeding populations of different ripeness groups and vegetation lengths. The studied amaranth species were A. caudatus L., A. mantegazzianus Passer., A. hybridus L., and A. hypochondriacus L. It was found that the enzymatic activity of malate dehydrogenase ensured the glyoxylate cycle functioning. The malate dehydrogenase activity varied depending on the enzyme accumulation during the initial stages of amaranth plant development. Isoenzyme analysis revealed two isoforms of malate dehydrogenase. It was noted that, in the studied amaranth species, the F-band was intrinsic to the early ripening forms, while the late-ripening carried the S-band. In addition, we detected the malate dehydrogenase polymorphism and differences in the enzymatic activity and demonstrated that it was possible to use them as a biochemical marker in breeding.

Authentic Research In A Large Laboratory Course: Studies of Metabolic Channeling Properties With Malate Dehydrogenase (MDH) Isoforms In Trypanosoma brucei

We are using a Course-Based Undergraduate Research Experience (CURE) model to provide opportunities for upper level Biochemistry and Molecular Biology (BMB) majors to gain exposure to laboratory research, and connect to the protein-centric MDH CURE community (MCC) of faculty from many institutions. Initially, students perform a four-week “boot camp” of common laboratory techniques and then work, as teams, on authentic research projects. We are interested in metabolic regulation in Trypanosoma brucei, the protozoan parasite that causes African sleeping sickness, this unicellular eukaryote is dependent on metabolic regulation for successful colonization of the Tsetse fly and subsequent infection of mammalian hosts. Growth within the fly utilizes oxidative phosphorylation of amino acids, whereas growth in the mammalian bloodstream is strictly glycolytic. Three MDH isoforms exist in T. brucei, localized to mitochondria, cytoplasm and glycosomes, respectively. We are interested in determining if any of the T. brucei MDH isoforms interact with other metabolic enzymes as appropriate to promote the proper metabolic program. Reports from other species some MDH isoforms form specific interactions with these enzymes to promote high flux of intermediates via substrate channeling. We have used student projects to explore interactions between T. brucei Malate dehydrogenase (MDH) and enzymes that act in adjacent steps in metabolic pathways of mitochondria or in cytoplasm. Students have generated clones and optimized recombinant expression of MDH, citrate synthase (CS) and fumarate hydratase (FH) isoforms. While some student teams design projects de novo, others choose to continue a project from a previous semester. Student teams have conducted expression studies, interaction assays and enzymatic analyses to characterize these enzymes. Students give presentations, a final poster and write and revise reports and a research proposal. Students also share their ideas and projects with other members of MCC, a consortium of faculty working on similar projects with other organisms. MCC CUREs have been shown to support student learning, retention and sense of ownership of research, and MCC provides a wealth of opportunities for comparative studies and to offer faculty and students access to scientific information and community beyond the students and instructors in the course, assisting development of hypotheses and sharing of reagents.

Three cytosolic NAD-malate dehydrogenase isoforms of Arabidopsis thaliana: on the crossroad between energy fluxes and redox signaling.

In yeast and animal cells, mitochondrial disturbances resulting from imbalances in the respiratory chain require malate dehydrogenase (MDH) activities for re-directing fluxes of reducing equivalents. In plants, in addition to mitochondria, plastids use malate valves to counterbalance and maintain redox-homeostasis. Arabidopsis expresses three cytosolic MDH isoforms, namely cyMDH1, cyMDH2, and cyMDH3, the latter possessing an N-terminal extension carrying a unique cysteine residue C2. In this study, redox-effects on activity and structure of all three cyMDH isoforms were analyzed in vitro. cyMDH1 and cyMDH2 were reversibly inactivated by diamide treatment, accompanied by dimerization via disulfide-bridge formation. In contrast, cyMDH3 forms dimers and higher oligomers upon oxidation, but its low specific activity is redox-independent. In the presence of glutathione, cyMDH1 and cyMDH2 are protected from dimerization and inactivation. In contrast, cyMDH3 still dimerizes but does not form oligomers any longer. From analyses of single and double cysteine mutants and structural modeling of cyMDH3, we conclude that the presence of C2 and C336 allows for multiple cross-links in the higher molecular mass complexes comprising disulfides within the dimer as well as between monomers of two different dimers. Furthermore, nuclear localization of cyMDH isoforms was significantly increased under oxidizing conditions in isolated Arabidopsis protoplasts, in particular of isoform cyMDH3. The unique cyMDH3 C2–C2-linked dimer is, therefore, a good candidate as a redox-sensor taking over moonlighting functions upon disturbances of energy metabolism, as shown previously for the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) where oxidative modification of the sensitive catalytic cysteine residues induces nuclear translocation.

Providing Authentic Research Opportunities In A Large Laboratory Course By Studying Metabolic Channeling Properties Of Malate Dehydrogenase Isoforms In Trypanosoma Brucei

We are using a Course‐Based Undergraduate Research Experience (CURE) model to provide opportunities for Biochemistry and Molecular Biology (BMB) majors to gain exposure to laboratory research. Upper class undergraduates perform a four‐week “boot camp” of common laboratory techniques and then work, as teams, on projects related to a faculty member’s research. Proejcts focused on exploring interactions between Malate dehydrogenase (MDH) with citrate synthase or fumarate hydratase, adjacent enzymes in the tricarboxylic acid (TCA) cycle. Recent studies from some plant and mammalian species suggest that some MDH isoforms form specific interactions with these enzymes to promote high flux of intermediates via substrate channeling. We are interested in metabolic regulation in Trypanosoma brucei, the protozoan parasite that causes African sleeping sickness, this unicellular eukaryote is dependent on metabolic regulation for successful colonization of the Tsetse fly and subsequent infection of mammalian hosts. Growth within the fly utilizes oxidative phosphorylation of amino acids, whereas growth in the mammalian bloodstream is strictly glycolytic. Three MDH isoforms exist in T. brucei, localized to mitochondria, cytoplasm and glycosomes, respectively. We are interested in determining if any of the T. brucei MDH isoforms interact with other metabolic enzymes as appropriate to promote the proper metabolic program. We have used student projects to generate clones for recombinant expression of all three trypanosome MDHs, citrate synthase (CS) and both fumarate hydratase (FH) isoforms. While some student teams design projects de novo, others choose to continue a project from a previous semester. Student teams have conducted expression studies, interaction assays and enzymatic analyses to characterize these enzymes. Students give presentations, a final poster and write and revise reports and a research proposal. Student also share their ideas and projects with other members of a consortium of faculty from other institutions in the protein‐centric MDH CURE community (MCC), working on similar projects with other organisms. MCC provides a wealth of possibilities for comparative studies and to offer faculty and students access to scientific information and community beyond the students and instructors in the course, assisting development of hypotheses and sharing of reagents.

Analysis of protein‐protein interactions between isoforms of malate dehydrogenase and citrate synthase

Sequential enzymes involved in a metabolic pathway are often found in specific and dynamic protein‐protein interactions. The assembly of proteins into this metabolon allow the efficient transfer of substrate between adjacent proteins increasing the throughput and decreasing the potential for side metabolic reactions to occur between competing enzymes. One such pairing is the malate dehydrogenase (MDH) and citrate synthase (CS). In the forward direction of the tricarboxylic cycle, the oxidation of malate to OAA catalyzed by MDH is thermodynamically unfavorable. Coupling and potentially direct shuttling of product‐substrate affords the cycle to continue. The interaction site between mitochondrial MDH and CS has been partially identified via computational docking and cross‐linking studies. However, the domains and key residues of interaction and their impact on the actions and affinity of various isoforms of MDH and CS have not been identified. Using two biophysical techniques, protein thermal melts and surface plasminogen resonance, we have found both mitochondrial and cytosolic human MDH interact with CS (mitochondrial 3 fold greater than cytosolic), while plant MDH shows no interaction. Traditional pulldowns were used in addition to biophysical techniques. Using molecular crowding agents, we have identified the affinity and stoichiometry of interactions. This will allow us to probe the structures for key residues of interaction in the future.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Authentic research in the teaching laboratory at a large university: comparative studies of malate dehydrogenase isoforms in trypanosomes

Offering authentic research experiences to all Biochemistry and Molecular Biology (BMB) majors at a large university can be challenging. BMB at UMass Amherst has developed a series of Course‐Based Undergraduate Research experiences (CUREs), required for all majors. One of these focuses on studying Malate dehydrogenase (MDH) isoforms in Trypanosoma brucei, a protozoan parasite that causes African sleeping sickness. This organism utilizes oxidative phosphorylation of amino acids during growth in its Tsetse fly host, but is strictly glycolytic during growth in the mammalian bloodstream, thus understanding metabolic regulation in T. brucei is important to elucidating how the parasite proliferates in the host. T. brucei has three functional MDH isoforms localized to mitochondria, cytoplasm or glycosomes, respectively. MDH is an important enzyme that catalyzes the oxidation of oxaloacetate to malate, concomitant with the reduction of NAD+ to NADH. It highly conserved and important not only as part of the citric acid cycle reactions but also as part of gluconeogenesis and of the malate aspartate shuttle used to maintain redox balance. The wealth of structural and functional information about MDH from model organisms is used to inform studies of the orthologs from a range of evolutionarily distinct organisms, currently being conducted by a consortium of collaborating faculty from various colleges and universities out as part of the protein‐centric MDH CURE community (MCC). This collaboration gives faculty and students access to community beyond the students and instructors in the course. We have generated clones for recombinant expression of all three trypanosome MDHs and have begun kinetic analyses on the cytoplasmic and glycosomal isoforms, and are optimizing expression levels for the mitochondrial form. Students can design a projects de novo or choose to continue a project from students in a previous semester. During the term students give a presentation and have many writing assignments, with opportunities for peer review and revision, all teams present their results in a department‐wide poster session at the end of the semester. Projects a have included comparisons between recombinant forms with C‐ or N‐terminal tags and between different isoforms, or orthologs of different organisms, function at different temperature, pH or ionic strength. These studies provide tools for further analysis of biochemical and kinetic properties of the T. brucei enzymes and to perform comparative analyses with well characterized mammalian and plant MDH enzymes.Support or Funding InformationNSF IUSE “An Interdisciplinary Faculty Community Using a Protein‐focused Course Based Undergraduate Research Experience (CURE) to Improve Student Learning,”This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Self-protection of cytosolic malate dehydrogenase against oxidative stress in Arabidopsis.

Plant malate dehydrogenase (MDH) isoforms are found in different cell compartments and function in key metabolic pathways. It is well known that the chloroplastic NADP-dependent MDH activities are strictly redox regulated and controlled by light. However, redox dependence of other NAD-dependent MDH isoforms have been less studied. Here, we show by in vitro biochemical characterization that the major cytosolic MDH isoform (cytMDH1) is sensitive to H2O2 through sulfur oxidation of cysteines and methionines. CytMDH1 oxidation affects the kinetics, secondary structure, and thermodynamic stability of cytMDH1. Moreover, MS analyses and comparison of crystal structures between the reduced and H2O2-treated cytMDH1 further show that thioredoxin-reversible homodimerization of cytMDH1 through Cys330 disulfide formation protects the protein from overoxidation. Consistently, we found that cytosolic thioredoxins interact specifically with cytMDH in a yeast two-hybrid system. Importantly, we also show that cytosolic and chloroplastic, but not mitochondrial NAD-MDH activities are sensitive to H2O2 stress in Arabidopsis. NAD-MDH activities decreased both in a catalase2 mutant and in an NADP-thioredoxin reductase mutant, emphasizing the importance of the thioredoxin-reducing system to protect MDH from oxidation in vivo. We propose that the redox switch of the MDH activity contributes to adapt the cell metabolism to environmental constraints.

MDH2 Stimulated by Estrogen-GPR30 Pathway Down-Regulated PTEN Expression Promoting the Proliferation and Invasion of Cells in Endometrial Cancer

PURPOSE: The relationship between endometrial carcinoma and cellular metabolism is unknown. In endometrial cancer, mutation rate of PTEN has been reported very high. Malate dehydrogenase 2 (MDH2) is one of the isoforms of malate dehydrogenase, which is involved in citric acid cycle in mitochondria. Our study aimed to investigate the role MDH2 played in PTEN-regulated endometrial carcinoma. METHODS: To reveal the expression of MDH2 and the co-localization of PTEN and MDH2, immunohistochemistry and immunofluorescent staining were used. Western blot, Real-time PCR, RNA interference and overexpression plasmid DNA transfection were performed to investigate the relationship between PTEN and MDH2 as well as the impact of E2 on the expression of PTEN and MDH2, while CCK8, transwell and flow cytometric analysis were carried out to evaluate the proliferation, migration and invasion and apoptosis of endometrial carcinoma cell lines. RESULTS: Our results demonstrated that as a metabolism related enzyme, MDH2 was overexpressed in endometrial carcinoma tissues and related to the grade of the cancer (P=.038). Western blot, Real-time PCR and immunofluorescent staining revealed MDH2 inhibited the expression of PTEN and was co-localized with PTEN in the cytoplasm of endometrial carcinoma. Proliferation, transwell and apoptosis assay suggested that MDH2 enhanced the proliferation, migration and invasion but inhibited the apoptosis of endometrial cancer cell line through suppressing PTEN. Furthermore, E2 inhibited the expression level of PTEN but enhanced MDH2 via GPR30. CONCLUSIONS: Our study demonstrated that MDH2, stimulated by estrogen, was involved in the development of PTEN-regulated endometrial carcinoma through GPR30-related pathway.

Atividade da malato desidrogenase muscular de Astyanax bimaculatus da bacia hidrográfica do rio Una como biomarcadora de impacto ambiental

The enzymatic activity of malate dehydrogenase (MDH) and its isoforms may vary in fishes according their living environment. The aim of this work was to investigate the activity of MDH in the muscle tissue of Astyanax bimaculatus (locally called as lambari do rabo amarelo), collected at three different points (P1, P2 and P3) in the Una River Basin (Taubaté, SP), to evaluate it as a candidate biomarker for environmental change. Cytosolic and mitochondrial isoforms of MDH were extracted from muscles. The results indicated that the total enzyme activity was 1.8 and 2.5 times higher in A. bimaculaus at P2, in comparison to specimens collected at P1 and P3, respectively. Seven MDH isoforms were detected in the muscle tissue (MDH-1 to MDH-7). MDH-1 levels of specimens collected at P3 were 1.9 and 2.4 times higher than those from P1 and P2, respectively. However, the levels of MDH-5, -6 and -7 of P3 specimens were significantly lower than P1 ones. These results are possibly related to water quality, considering that P3 is the most polluted among the three sampling sites. The muscle MDH activity of A. bimaculatus was modulated by the environment. Therefore, it can be concluded that this enzyme could be used as a biomarker of environmental change of the hydrographic basin of a neotropical region, where this species occurs.

Physicochemical and catalytic properties of NAD+- dependent malate dehydrogenase isoforms from maize mesophyll

Malate dehyrogenase isoforms (46- and 70-fold purifications) with specific activities of the 640 and 990 U/mg protein were obtained in an electrophoretically homogeneous state from maize mesophyll. The physicochemical and catalytic properties of these isoforms were studied. The molecular weight and the Michaelis constants were determined; the effect of hydrogen ions on the forward and reverse MDH reaction was studied. The results of SDS-PAGE demonstrated that malate dehydrogenase isoforms have an oligomeric structure comprised of identical subunits. The first isoform with a molecular weight of 126.58 kDa is tetramer, and the second isoform with a molecular weight of 63.3 is dimer.

Tuning of Redox Regulatory Mechanisms, Reactive Oxygen Species and Redox Homeostasis under Salinity Stress.

Soil salinity is a crucial environmental constraint which limits biomass production at many sites on a global scale. Saline growth conditions cause osmotic and ionic imbalances, oxidative stress and perturb metabolism, e.g., the photosynthetic electron flow. The plant ability to tolerate salinity is determined by multiple biochemical and physiological mechanisms protecting cell functions, in particular by regulating proper water relations and maintaining ion homeostasis. Redox homeostasis is a fundamental cell property. Its regulation includes control of reactive oxygen species (ROS) generation, sensing deviation from and readjustment of the cellular redox state. All these redox related functions have been recognized as decisive factors in salinity acclimation and adaptation. This review focuses on the core response of plants to overcome the challenges of salinity stress through regulation of ROS generation and detoxification systems and to maintain redox homeostasis. Emphasis is given to the role of NADH oxidase (RBOH), alternative oxidase (AOX), the plastid terminal oxidase (PTOX) and the malate valve with the malate dehydrogenase isoforms under salt stress. Overwhelming evidence assigns an essential auxiliary function of ROS and redox homeostasis to salinity acclimation of plants.

Isoformes of Malate Dehydrogenase from Rhodovulum Steppense A-20s Grown Chemotrophically under Aerobic Conditions

Three malate dehydrogenase isoforms (65-, 60-, and 71-fold purifications) with specific activities of 4.23, 3.88, and 4.56 U/mg protein were obtained in an electrophoretically homogenous state from Rhodovulum steppense bacteria strain A-20s chemotropically grown under aerobic conditions. The physicochemical and kinetic properties of malate dehydrogenase isoforms were determined. The molecular weight and the Michaelis constants were determined; the effect of hydrogen ions on the forward and reverse MDH reaction was studied. The results of the study demonstrated that the enzyme consists of subunits; the molecular weight of subunits was determined by SDS-PAGE.

Enhanced cytosolic NADP-ME2 activity in A. thaliana affects plant development, stress tolerance and specific diurnal and nocturnal cellular processes

Arabidopsis thaliana has four NADP-dependent malic enzymes (NADP-ME 1-4) for reversible malate decarboxylation, with NADP-ME2 being the only cytosolic isoform ubiquitously expressed and responsible for most of the total activity. In this work, we further investigated its physiological function by characterizing Arabidopsis plants over-expressing NADP-ME2 constitutively. In comparison to wild type, these plants exhibited reduced rosette and root sizes, delayed flowering time and increased sensitivity to mannitol and polyethylene glycol. The increased NADP-ME2 activity led to a decreased expression of other ME and malate dehydrogenase isoforms and generated a redox imbalance with opposite characteristics depending on the time point of the day analyzed. The over-expressing plants also presented a higher content of C4 organic acids and sugars under normal growth conditions. However, the accumulation of these metabolites in the over-expressing plants was substantially less pronounced after osmotic stress exposure compared to wild type. Also, a lower level of several amino acids and osmoprotector compounds was observed in transgenic plants. Thus, the gain of NADP-ME2 expression has profound consequences in the modulation of primary metabolism in A. thaliana, which reflect the relevance of this enzyme and its substrates and products in plant homeostasis.

Molecular mechanisms of the non-coenzyme action of thiamin in brain: biochemical, structural and pathway analysis.

Thiamin (vitamin B1) is a pharmacological agent boosting central metabolism through the action of the coenzyme thiamin diphosphate (ThDP). However, positive effects, including improved cognition, of high thiamin doses in neurodegeneration may be observed without increased ThDP or ThDP-dependent enzymes in brain. Here, we determine protein partners and metabolic pathways where thiamin acts beyond its coenzyme role. Malate dehydrogenase, glutamate dehydrogenase and pyridoxal kinase were identified as abundant proteins binding to thiamin- or thiazolium-modified sorbents. Kinetic studies, supported by structural analysis, revealed allosteric regulation of these proteins by thiamin and/or its derivatives. Thiamin triphosphate and adenylated thiamin triphosphate activate glutamate dehydrogenase. Thiamin and ThDP regulate malate dehydrogenase isoforms and pyridoxal kinase. Thiamin regulation of enzymes related to malate-aspartate shuttle may impact on malate/citrate exchange, responsible for exporting acetyl residues from mitochondria. Indeed, bioinformatic analyses found an association between thiamin- and thiazolium-binding proteins and the term acetylation. Our interdisciplinary study shows that thiamin is not only a coenzyme for acetyl-CoA production, but also an allosteric regulator of acetyl-CoA metabolism including regulatory acetylation of proteins and acetylcholine biosynthesis. Moreover, thiamin action in neurodegeneration may also involve neurodegeneration-related 14-3-3, DJ-1 and β-amyloid precursor proteins identified among the thiamin- and/or thiazolium-binding proteins.

On the consequences of aluminium stress in rye: repression of two mitochondrial malate dehydrogenase mRNAs.

Plants have developed several external and internal aluminium (Al) tolerance mechanisms. The external mechanism best characterised is the exudation of organic acids induced by Al. Rye (Secale cereale L.), one of the most Al-tolerant cereal crops, secretes both citrate and malate from its roots in response to Al. However, the role of malate dehydrogenase (MDH) genes in Al-induced stress has not been studied in rye. We have isolated the ScMDH1 and ScMDH2 genes, encoding two different mitochondrial MDH isozymes, in three Al-tolerant rye cultivars (Ailés, Imperial and Petkus) and one sensitive inbred rye line (Riodeva). These genes, which have seven exons and six introns, were located on the 1R (ScMDH1) and 3RL (ScMDH2) chromosomes. Exon 1 of ScMDH1 and exon 7 of ScMDH2 were the most variable among the different ryes. The hypothetical proteins encoded by these genes were classified as putative mitochondrial MDH isoforms. The phylogenetic relationships obtained using both cDNA and protein sequences indicated that the ScMDH1 and ScMDH2 proteins are orthologous to mitochondrial MDH1 and MDH2 proteins of different Poaceae species. The expression studies of the ScMDH1 and ScMDH2 genes indicate that it is more intense in roots than in leaves. Moreover, the amount of their corresponding mRNAs in roots from plants treated and not treated with Al was higher in the tolerant cultivar Petkus than in the sensitive inbred line Riodeva. In addition, ScMDH1 and ScMDH2 mRNA levels decreased in response to Al stress (repressive behaviour) in the roots of both the tolerant Petkus and the sensitive line Riodeva.

Inactivation of mitochondrial aspartate aminotransferase contributes to the respiratory deficit of yeast frataxin-deficient cells

Friedreich's ataxia is a hereditary neurodegenerative disease caused by reduced expression of mitochondrial frataxin. Frataxin deficiency causes impairment in respiratory capacity, disruption of iron homoeostasis and hypersensitivity to oxidants. Although the redox properties of NAD (NAD+ and NADH) are essential for energy metabolism, only few results are available concerning homoeostasis of these nucleotides in frataxin-deficient cells. In the present study, we show that the malate-aspartate NADH shuttle is impaired in Saccharomyces cerevisiae frataxin-deficient cells (Δyfh1) due to decreased activity of cytosolic and mitochondrial isoforms of malate dehydrogenase and to complete inactivation of the mitochondrial aspartate aminotransferase (Aat1). A considerable decrease in the amount of mitochondrial acetylated proteins was observed in the Δyfh1 mutant compared with wild-type. Aat1 is acetylated in wild-type mitochondria and deacetylated in Δyfh1 mitochondria suggesting that inactivation could be due to this post-translational modification. Mutants deficient in iron-sulfur cluster assembly or lacking mitochondrial DNA also showed decreased activity of Aat1, suggesting that Aat1 inactivation was a secondary phenotype in Δyfh1 cells. Interestingly, deletion of the AAT1 gene in a wild-type strain caused respiratory deficiency and disruption of iron homoeostasis without any sensitivity to oxidative stress. Our results show that secondary inactivation of Aat1 contributes to the amplification of the respiratory defect observed in Δyfh1 cells. Further implication of mitochondrial protein deacetylation in the physiology of frataxin-deficient cells is anticipated.

Mechanism of malate dehydrogenase isoform formation in Sphaerotilus natans D-507 under different cultivation conditions

Electrophoretically homogenous preparations of malate dehydrogenase (MDH) isoforms of the bacteria Sphaerotilus natans D-507 with specific activity 7.46 U/mg and 5.74 U/mg with respect to protein concentration have been obtained. The dimeric isoform of the enzyme was shown to function under organotrophic growth conditions, whereas the tetrameric isoform was induced under mixotrophic cultivation conditions. PCR-analysis revealed a single gene encoding the malate dehydrogenase molecule. The topography of the MDH isoform surface was studied by atomic-force microscopy, and a 3D-structure of the enzyme was obtained. Spectraphotometric analysis data allowed us to suggest that stabilization of the tetrameric form of MDH is due to additional bounds implicated in the quaternary structure formation.

Proteomic changes in the heart of diet-induced pre-diabetic mice

The development of type 2 diabetes (T2D) is strongly associated with obesity. In humans, T2D increases the risk for end organ complications. Among these, heart disease has been ranked as the leading cause of death. We used a proteomic methodology to test the hypothesis that a pre-diabetic state generated by high-fat diet leads to changes in proteins related to heart function and structure. Over 300 protein spots were resolved by two-dimensional gel electrophoresis (2-DE). Fifteen protein spots were found to be altered (7 decreased and 8 increased) in pre-diabetic hearts. The protein spots were then identified by mass spectrometry and immunoblots. Among the decreased proteins, 3 are involved in heart structure (one isoform of desmin, troponin T2 and α-cardiac actin), 3 are involved in energy metabolism (mitochondrial ATP synthase β subunit, adenylate kinase and creatine kinase) and one is a component of the citric acid cycle (isocitrate dehydrogenase 3). In contrast, proteins involved in fatty acid oxidation (two isoforms of peroxisomal enoyl-CoA hydratase) and the citric acid cycle (three isoforms of malate dehydrogenase) were increased in pre-diabetic hearts. The results suggest that changes in the levels of several heart proteins may have implications in the development of the cardiac phenotype associated to T2D.

Ethanol‐induced redox imbalance in rat kidneys

This study reports the effects of long-term ethanol consumption on kidney redox status, in terms of enzymatic mechanisms involved in regulating the cytosolic [NADH]/[NAD(+) ] balance. Wistar rats were treated with ethanol (2 g/kg body weight/24 h) via intragastric intubation for 10 and 30 weeks, respectively. Ethanol administration induced an enhancement of alcohol dehydrogenase activities and affected the capacity of the kidney to prevent NADH accumulation in the cytosol. After 10 weeks, the excess of NADH was balanced by increased activities of malate dehydrogenase and aspartate transaminase. In the event of a longer period of ethanol intake, the kidney was not able to balance the NADH excess, even though an increase in malate dehydrogenase, lactate dehydrogenase, aspartate transaminase, and alanine transaminase activities was noted. The electrophoretic analysis of alcohol dehydrogenase, lactate dehydrogenase, and malate dehydrogenase isoforms revealed differences between control and ethanol-treated animals. The results suggest that rat kidneys have a multicomponent metabolic response to the same daily dose of ethanol that functions to maintain the redox status and which varies with the length of the administration period.